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Long undecoded transcript isoforms (LUTIs) represent a class of non-canonical mRNAs that downregulate gene expression through the combined act of transcriptional and translational repression. While single gene studies revealed important aspects of LUTI-based repression, how these features affect gene regulation on a global scale is unknown. Using transcript leader and direct RNA sequencing, here, we identify 74 LUTI candidates that are specifically induced in meiotic prophase. Translational repression of these candidates appears to be ubiquitous and is dependent on upstream open reading frames. However, LUTI-based transcriptional repression is variable. In only 50% of the cases, LUTI transcription causes downregulation of the protein-coding transcript isoform. Higher LUTI expression, enrichment of histone 3 lysine 36 trimethylation, and changes in nucleosome position are the strongest predictors of LUTI-based transcriptional repression. We conclude that LUTIs downregulate gene expression in a manner that integrates translational repression, chromatin state changes, and the magnitude of LUTI expression. [Display omitted] • Long-read RNA-seq reveals 5′-extended mRNAs as candidate LUTIs in yeast meiosis • Majority of the LUTIs are regulated by a cell-fate-instructive transcription factor • LUTI-based translational repression depends on uORFs • Chromatin changes and LUTI expression predict LUTI-based transcriptional repression Tresenrider et al. conducted a genome-wide study to dissect an unconventional gene regulatory mechanism whereby transcription factors could function as repressors by inducing non-canonical mRNAs called LUTIs. Their findings reveal that LUTI-based gene repression depends on a combination of factors, including translational repression, chromatin state changes, and LUTI expression. [ABSTRACT FROM AUTHOR]

LUTIs (Long Undecoded Transcript Isoforms) are 5'-extended and poorly translated mRNAs that can downregulate transcription from promoters more proximal to a gene's coding sequence (CDS). In this protocol, polyA RNA is extracted from budding yeast cells undergoing highly synchronized meiosis. Using a combination of long-read direct RNA sequencing and transcript leader sequencing (TL-seq), meiosis-specific LUTIs are systematically identified. Following identification, TL-seq is used to quantify the abundance of both LUTI and the more canonical gene-proximal (PROX) transcripts. For complete details on the use and execution of this protocol, please refer to Tresenrider et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

Transcription through noncoding regions of the genome is pervasive. How these transcription events regulate gene expression remains poorly understood. Here, we report that, in S. cerevisiae, the levels of transcription through a noncoding region, IRT2, located upstream in the promoter of the inducer of meiosis, IME1, regulate opposing chromatin and transcription states. At low levels, the act of IRT2 transcription promotes histone exchange, delivering acetylated histone H3 lysine 56 to chromatin locally. The subsequent open chromatin state directs transcription factor recruitment and induces downstream transcription to repress the IME1 promoter and meiotic entry. Conversely, increasing transcription turns IRT2 into a repressor by promoting transcription-coupled chromatin assembly. The two opposing functions of IRT2 transcription shape a regulatory circuit, which ensures a robust cell-type-specific control of IME1 expression and yeast meiosis. Our data illustrate how intergenic transcription levels are key to controlling local chromatin state, gene expression, and cell fate outcomes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Many active eukaryotic gene promoters exhibit divergent noncoding transcription, but the mechanisms restricting expression of these transcripts are not well understood. Here, we demonstrate how a sequence-specific transcription factor represses divergent noncoding transcription at highly expressed genes in yeast. We find that depletion of the transcription factor Rap1 induces noncoding transcription in a large fraction of Rap1-regulated gene promoters. Specifically, Rap1 prevents transcription initiation at cryptic promoters near its binding sites, which is uncoupled from transcription regulation in the protein-coding direction. We further provide evidence that Rap1 acts independently of previously described chromatin-based mechanisms to repress cryptic or divergent transcription. Finally, we show that divergent transcription in the absence of Rap1 is elicited by the RSC chromatin remodeler. We propose that a sequence-specific transcription factor limits access of basal transcription machinery to regulatory elements and adjacent sequences that act as divergent cryptic promoters, thereby providing directionality toward productive transcription., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)